Broad detection of dengue virus serotypes

ABSTRACT

Processes for detecting Dengue virus (DENV) nucleic acid in a sample are provided including producing an amplification product by amplifying DENV nucleotide sequence and detection of an amplification by hybridization of a probe or other technique. The processes use primers or probes with introduced mutations and or degenerate bases that provide excellent superiority in detection and serotyping of DENV in a sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application depends from and claims priority to U.S. PatentApplication No. 61/554,126 filed Nov. 1, 2011, the entire contents ofwhich are incorporated herein by reference.

GOVERNMENT INTEREST

The invention described herein may be manufactured, used, and licensedby or for the United States Government.

FIELD OF THE INVENTION

This invention relates generally to processes for detection of foreignorganisms in fluid samples. More specifically, the invention relates toselective detection of dengue virus (DENV) in biological or other fluidmedia. Processes are described for rapid and sensitive detection andsubtyping (serotyping) of DENV in biological samples and quantificationthereof. Diagnostic kits are provided for detection of DENV serotypes inblood (serum or plasma) samples from patients with symptoms of dengueillness in a clinical, laboratory, or field setting.

BACKGROUND OF THE INVENTION

Dengue virus (DENV) is the cause of dengue illness (dengue fever, denguehemorrhagic fever and dengue shock syndrome), which is reported inhundreds of thousands of people each year. Dengue hemorrhagic fever isfatal in about 0.5% of cases. DENVs are a group of four closely relatedarboviruses represented by serotypes DENV-1, DENV-2, DENV-3, and DENV-4.In areas of high virus presence, all four of the DENV serotypes may becirculating simultaneously.

The four DENV serotypes are antigenically distinct, but relatedserologically. Thus, infection by one serotype does not provideprotection from subsequent infections by other serotypes. Studiesdemonstrated that host developed antibodies directed to the firstinfecting serotype may have some affinity for a second infectingserotype and lead to enhancement of the ability of the second virus toinfect the host.

Patients with dengue usually seek medical attention during the first 5days of illness; when the virus is present in the blood and IgMantibodies are not yet detectable. Therefore, diagnostic tests highlyrely on the ability to detect virus components such as viral RNA.

Identification of DENV through serological methods is complicated due toextensive cross-reactivity between flaviviruses. The level ofcirculating IgM is not uniform based on the type of infection. Forexample, infection by a second DENV serotype may show little to no IgMresponse, and its presence may be masked by circulating IgM from eitherprior DENV infection, or from infection by another flavivirus. Thus, inareas with high or epidemic transmission of multiple flaviviruses,identification of DENV may not be possible by serological methods.

Within each DENV serotype, there are 4-6 genotypes, which representlineages that may differ from one another in approximately 5-10% oftheir genomes.

Thus, there is a need for compositions and methods for the specificdetection of DENV in fluidic samples such as whole blood, plasma, orserum early during disease presentation that does not rely on antibodyserology, yet is sufficiently specific enough to broadly detect multipleDENV serotypes.

SUMMARY OF THE INVENTION

The following summary of the invention is provided to facilitate anunderstanding of some of the innovative features unique to the presentinvention and is not intended to be a full description. A fullappreciation of the various aspects of the invention can be gained bytaking the entire specification, claims, drawings, and abstract as awhole.

Compositions and methods are provided for superior detection of DENV ina sample such as a fluidic sample. These methods capitalize on one ormore primers or probes suitable for use with an assay systemcapitalizing on the polymerase chain reaction that allow detection ofall known viruses in a specific serotype. As such, the methods andcompositions provided may be used to provide superior confidence indetermining the presence or absence of a DENV virus in a sample ineither a singleplex or multiplex assay format

Processes are provided for detecting DENV in a sample that includeproducing an amplification product by amplifying a nucleotide sequenceusing a forward primer that hybridizes to a first region within theDengue virus genome, and a reverse primer that hybridizes to a secondregion within the Dengue virus genome, under conditions suitable for apolymerase chain reaction, and optionally wherein at least one of theforward primer or reverse primer has one or more degenerate ornon-wild-type nucleotide substitutions; and detecting a first detectionsignal correlating to the presence of the amplification product todetect the Dengue virus in a sample. A first primer optionally includesthe sequence of SEQ ID NO: 1; SEQ ID NO: 4; SEQ ID NO: 7, SEQ ID NO: 10,or combinations of these primers are used. A reverse primer optionallyincludes the sequence of SEQ IN NO: 2; SEQ ID NO: 5; SEQ ID NO: 8, SEQID NO: 11, or combinations of these primers are used. A probe optionallyincludes the sequence of SEQ ID NO: 3; SEQ ID NO: 6; SEQ ID NO: 9, SEQID NO: 12, or combinations these probes are used.

Any suitable detection system can be used to detect an amplificationproduct, even in the absence of a probe. In the absence of a probe afirst detection signal is produced by the amplification product itselfor by a non-probe signal directed to the amplification product.Illustratively, an amplification product is detected by gelelectrophoresis, Southern blotting, liquid chromatography, massspectrometry, liquid chromatography/mass spectrometry, massspectrometry, static fluorescence, dynamic fluorescence, highperformance liquid chromatography, ultra-high performance liquidchromatography, enzyme-linked immunoadsorbent assay, real-time PCR,nucleotide sequencing, or combinations thereof.

The compositions and processes can be used to diagnose or confirm thediagnosis of the presence or absence of DENV in a subject or confirm thepresence or absence of a DENV in a sample, or for the preparation of acomposition, kit, or other device that may be used to diagnose orconfirm the diagnosis of the presence or absence of DENV in a subject ora sample.

The compositions and processes provided dramatically improve confidencein diagnosis or detection of DENV from one or more serotypes in asample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates DENV serotypes 1-4 specific detection as measured ingenome copy equivalents per mL of sample (GCE/mL) between virusdilutions in human serum and human plasma demonstrating FIG. 1A(singleplex) and 1B (multiplex) assay formats;

FIG. 2 represents the ability of the assay to detect DENV serotypesfollowing multiple freeze/thaw cycles.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The following description of particular embodiment(s) is merelyexemplary in nature and is in no way intended to limit the scope of theinvention, its application, or uses, which may, of course, vary. Theinvention is described with relation to the non-limiting definitions andterminology included herein. These definitions and terminology are notdesigned to function as a limitation on the scope or practice of theinvention but are presented for illustrative and descriptive purposesonly. While the processes are described as an order of individual stepsor using specific materials, it is appreciated that described steps ormaterials may be interchangeable such that the description of theinvention includes multiple parts or steps arranged in many ways as isreadily appreciated by one of skill in the art. While numerous elementsof the description are directed to primers of SEQ ID NO: 1, SEQ ID NO:2, and probe of SEQ ID NO: 3, it is appreciated that other primers orprobes are substitutable as is readily appreciated by one of skill inthe art as well as the inclusion of primers of SEQ ID NO: 1, SEQ ID NO:2, and probe of SEQ ID NO: 3 in a multiplex assay along with otherprimers and probes.

The invention has utility for the detection of DENV in a sample. As itis currently necessary to detect DENV in clinical specimens by thestandard serological methods, sensitive techniques such as RT-PCR mayprovide a more reliable diagnostic than other currently employed assaysystems. Prior attempts at designing nucleic acid based diagnosticmethods have met with limited success due to poor recognition ofmultiple DENV serotypes, inability to multiplex the assay, or lack ofsensitivity. Despite the failings of prior investigators, the inventorsidentified a new family of primers and probes suitable for single ormultiplexed real-time RT-PCR that are far superior to prior primer andprobe sequences in their ability to amplify and detect all four DENVserotypes from multiple members of the serotypes that have sequencevariations.

Compositions and methods are provided for the sensitive detection ofDENV in samples, such as biological or environmental samples, usingtechniques involving PCR. Primers are provided that amplify regions ofDENV genetic RNA with high specificity and broad DENV recognition thatare subsequently detectable, optionally by sensitive detection systems.

The following definitional terms are used throughout the specificationwithout regard to placement relative to these terms.

As used herein, the term “variant” defines either a naturally occurringgenetic mutant of DENV or a recombinantly prepared variation of theDENV. The term “variant” may also refer to either a naturally occurringvariation of a given encoded peptide or a recombinantly preparedvariation of a given encoded peptide or protein in which one or moreamino acid residues have been modified by amino acid substitution,addition, or deletion.

As used herein, the term “analog” in the context of a non-proteinaceousanalog defines a second organic or inorganic molecule that possesses asimilar or identical function as a first organic or inorganic moleculeand is structurally similar to the first organic or inorganic molecule.

As used herein, the term “derivative” in the context of anon-proteinaceous derivative defines a second organic or inorganicmolecule that is formed based upon the structure of a first organic orinorganic molecule. A derivative of an organic molecule includes, but isnot limited to, a molecule modified, e.g., by the addition or deletionof a hydroxyl, methyl, ethyl, carboxyl or amine group. An organicmolecule may also be esterified, alkylated and/or phosphorylated. Aderivative also defined as a degenerate base mimicking a C/T mix such asthat from Glen Research Corporation, Sterling, Va., illustrativelyLNA-dA or LNA-dT, or other nucleotide modification known in the art orotherwise.

As used herein, the term “mutant” defines the presence of mutations inthe nucleotide sequence of an organism as compared to a wild-typeorganism. A mutant is a variant.

The description of primers and probes to amplify and detect one or moretarget nucleic acid molecule is presented. In some embodiments, theprimers, probes, or both specifically include variants, analogues,derivatives, and mutants of the sequences presented herein. In someembodiments, the primers, probes, or both specifically exclude variants,analogues, derivatives, and mutants of the sequences taught herein orthe wild-type sequences.

As used herein, the term “hybridizes under stringent conditions”describes conditions for hybridization and washing under whichnucleotide sequences having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, base pair matches to eachother typically remain hybridized to each other. Illustrativehybridization conditions are described in, for example but not limitedto, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1 6.3.6; Basic Methods in Molecular Biology, ElsevierScience Publishing Co., Inc. N.Y. (1986), pp. 75 78, and 84 87; andMolecular Cloning, Cold Spring Harbor Laboratory, N.Y. (1982), pp. 387389, and are well known to those skilled in the art. A non-limitingexample of stringent hybridization conditions is hybridization in 6×sodium chloride/sodium citrate (SSC), 0.5% SDS at about 60° C. followedby one or more washes in 2×SSC, 0.5% SDS at room temperature. Anothernon-limiting example of stringent hybridization conditions ishybridization in 6×SSC at about 45° C. followed by one or more washes in0.2×SSC, 0.1% SDS at 50 to 65° C. Other stringent hybridizationconditions will be evident to one of ordinary skill in the art based ongeneral knowledge in the art as well as this specification.

An “isolated” or “purified” nucleotide or oligonucleotide sequence issubstantially free of cellular material or other contaminating proteinsfrom the cell or tissue source from which the nucleotide is derived, oris substantially free of chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of cellularmaterial” includes preparations of a nucleotide/oligonucleotide in whichthe nucleotide/oligonucleotide is separated from cellular components ofthe cells from which it is isolated or produced. Thus, anucleotide/oligonucleotide that is substantially free of cellularmaterial includes preparations of the nucleotide having less than about30%, 20%, 10%, 5%, 2.5%, or 1%, (by dry weight) of contaminatingmaterial. When nucleotide/oligonucleotide is produced by chemicalsynthesis, it is optionally substantially free of chemical precursors orother chemicals, i.e., it is separated from chemical precursors or otherchemicals which are involved in the synthesis of the molecule.Accordingly, such preparations of the nucleotide/oligonucleotide haveless than about 30%, 20%, 10%, or 5% (by dry weight) of chemicalprecursors or compounds other than the nucleotide/oligonucleotide ofinterest. In some embodiments of the present invention, anucleotide/oligonucleotide is isolated or purified. This terms“isolated” or “purified” are exclusive of a nucleic acid that is amember of a library that has not been purified away from other libraryclones containing other nucleic acid molecules.

As used herein, the term “sample” is a portion of a larger source. Asample is optionally solid, gaseous, or fluidic. A sample isillustratively an environmental or biological sample. An environmentalsample is illustratively, but not limited to, water, sewage, soil, orair. A “biological sample” is as sample obtained from a biologicalorganism, a tissue, cell, cell culture medium, or any medium suitablefor mimicking biological conditions. Non-limiting examples include,saliva, gingival secretions, cerebrospinal fluid, gastrointestinalfluid, mucous, urogenital secretions, synovial fluid, blood, serum,plasma, urine, cystic fluid, lymph fluid, ascites, pleural effusion,interstitial fluid, intracellular fluid, ocular fluids, seminal fluid,mammary secretions, and vitreal fluid, and nasal secretions, throat ornasal materials. In some embodiments, target agents are contained in:serum; plasma; whole blood; feces; urine; throat fluid; nasopharyngealfluid; or other respiratory fluid.

As used herein, the term “medium” refers to any liquid or fluid samplein the presence or absence of a virus. A medium is illustratively asolid sample that has been suspended, solubilized, or otherwise combinedwith fluid to form a fluidic sample. Non-limiting examples includebuffered saline solution, cell culture medium, acetonitrile,trifluoroacetic acid, combinations thereof, or any other fluidrecognized in the art as suitable for combination with virus or othercells, or for dilution of a biological sample or amplification productfor analysis.

To determine the percent identity of two nucleic acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the sequence of a first amino acid or nucleic acidsequence for optimal alignment with a second amino acid or nucleic acidsequence). The nucleotides at corresponding nucleotide positions arethen compared. When a position in the first sequence is occupied by thesame nucleotide as the corresponding position in the second sequence,then the molecules are identical at that position. The percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences (i.e., % identity=number of identicaloverlapping positions/total number of positions .times.100%). In someembodiments, the two sequences are the same length.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul, 1990, PNAS 87:2264 2268, modifiedas in Karlin and Altschul, 1993, PNAS. 90:5873 5877. Such an algorithmis incorporated into the NBLAST and XBLAST programs of Altschul et al.,1990, J. Mol. Biol. 215:403. BLAST nucleotide searches are performedwith the NBLAST nucleotide program parameters set, e.g., for score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecules of the present invention. BLAST protein searches areperformed with the XBLAST program parameters set. e.g., to score 50.wordlength=3 to obtain amino acid sequences homologous to a proteinmolecule of the present invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST are utilized as described in Altschulet al., 1997, Nucleic Acids Res. 25:3389 3402. Alternatively, PSI BLASTis used to perform an iterated search which detects distantrelationships between molecules (Id.). When utilizing BLAST, GappedBLAST, and PSI Blast programs, the default parameters of the respectiveprograms (e.g., of XBLAST and NBLAST) are used (see, e.g., the NCBIwebsite). Another non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, 1988, CABIOS 4:11 17. Such an algorithm is incorporated in theALIGN program (version 2.0) which is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 is used.

The percent identity between two sequences is determined usingtechniques similar to those described herein or otherwise known in theart, with or without allowing gaps. In calculating percent identity,typically only exact matches are counted.

As used herein, the terms “subject” and “patient” are synonymous andrefer to a human or non-human animal, optionally a mammal including ahuman, a non-primate such as cows, pigs, horses, goats, sheep, cats,dogs, avian species and rodents; and a non-human primate such asmonkeys, chimpanzees, and apes; and a human, also optionally denotedspecifically as a “human subject”.

Processes are described that provide a rapid, specific, and sensitiveassay for detection of DENV in a sample by amplifying one or morenucleotide sequences of the RNA genome of DENV by processes similar tothe polymerase chain reaction (PCR). Processes are similarly providedfor diagnosing the presence or absence of DENV infection in a subject.The presence of DENV detected in a sample from the subject diagnoses orconfirms a prior diagnosis of infection of the subject by DENV. Theabsence of DENV in a sample from a subject diagnoses the absence of aninfection of the subject by DENV.

An oligonucleotide forward primer with a nucleotide sequencecomplementary to a unique sequence in a DENV nucleotide sequence ishybridized to its complementary sequence and extended. A nucleotidesequence is complementary if it hybridizes under stringent conditions.Similarly, a reverse oligonucleotide primer complementary to a secondstrand of DENV RNA (or reverse transcribed DNA) is hybridized andextended. The amplification products of the forward and reverse primeroverlap in complementary sequence providing a uniform amplificationproduct from each pair of forward and reverse primers. This systemallows for amplification of specific nucleic acid sequences and issuitable for simultaneous or sequential detection systems.

The present invention relates to the use of the sequence information ofDENV for diagnostic or other detection processes. In particular, thepresent invention provides a process for detecting the presence orabsence of nucleic acid molecules of DENV, natural or artificialvariants, analogs, or derivatives thereof, in a sample. In someembodiments, processes involve obtaining a biological sample from one ormore of various sources and contacting the sample with a compound or anagent (e.g. primer or probe) capable of detecting a nucleic acidsequence of DENV, natural or artificial variants, analogs, orderivatives thereof, such that the presence of DENV, natural orartificial variants, analogs, or derivatives thereof, is detected in thesample. Optionally, infection by DENV is diagnosed by positivelydetecting one or more DENV serotypes in the sample. In some embodiments,the presence of DENV, natural or artificial variants, analogs, orderivatives thereof, is detected in the sample using a PCR reaction orreal-time polymerase chain reaction (RT-PCR) including primers that areconstructed based on a partial nucleotide sequence of the DENV organismwith the proviso that at least one of a probe, forward primer, orreverse primer houses a sequence that is not wild-type. The inventorsdiscovered that by introduction of non-wild-type nucleotides, andoptionally one or more positions of non-wild type degeneracy, the use ofthe primers or probes results in much greater detection affinity thanpreviously detectable by prior primer or probe sequences or combinationsof sequences. The locations of a non-wild type nucleotide or degeneracywas surprising in that no knowledge in the art suggests that thesepositions or type of substitution would alter a detection assay.

Primers must be designed to amplify target from the greatest number ofDENV strains while simultaneously preventing false positives. In anon-limiting embodiment, a forward primer designed to be successful forselective amplification in a PCR based assay such as in a RT-PCR processis illustratively 5′-CAAAAGGAAGTCGYGCAATA-3′ (SEQ ID NO: 1). As usedherein the nucleotide notation “Y” represents a degenerate purine thatis either adenine or guanine. The nucleotide notation “R” as used hereinrepresents a degenerate pyrimidine that is either thymine or cytosine.In some embodiments for the detection of DENV-1, a reverse primerdesigned to be successful for selective amplification in a PCR basedassay such as in a RT-PCR process is illustratively5′-CTGAGTGAATTCTCTCTGCTRAAC-3′ (SEQ ID NO: 2). In some embodiments, theprimers used in a process are the nucleic acid sequences of SEQ ID NOs:1and 2. As used herein, the term “amplify” is defined as producing one ormore copies of a target molecule, or a complement thereof. A nucleicacid such as DNA or RNA is amplified to produce one or moreamplification products. Illustratively, a forward primer and an optionalreverse primer are contacted with a target under conditions suitable fora polymerase chain reaction to produce an amplification product.

An agent for detecting DENV nucleic acid sequences is optionally alabeled nucleic acid probe capable of hybridizing to a portion of theDENV genome, or amplification products derived therefrom such as DNAproduced by reverse transcription of the RNA genome or a portionthereof. In some embodiments for the detection of DENV-1, the nucleicacid probe is a nucleic acid molecule of the nucleic acid sequence of5′-CATGTGGYTGGGAGCRCGC-3′ (SEQ ID NO: 3), which sufficientlyspecifically hybridizes under stringent conditions to a DENV nucleicacid sequence. A probe is optionally labeled with a fluorescent moleculesuch as a fluorescein (FAM) molecule and optionally a quencher such asthe black hole quencher BHQ1.

Primers and probes are provided for singleplex or multiplex detection ofone or more serotypes of DENV in a sample. Illustrative primers andprobes are provided in Table 1 where bold nucleotides representdegenerate base location or other non-wild type substitutions.

TABLE 1 Oligonucleotide SEQ ID NO: D1-F CDC DENV-1-4 Real Time RT-PCRCAAAAGGAAGTCGYGCAATA 1 D1-R CDC DENV-1-4 Real Time RT-PCRCTGAGTGAATTCTCTCTGCTRAAC 2 D1-Probe CDC DENV-1-4 Real Time RT-PCRCATGTGGYTGGGAGCRCGC 3 D2-F CDC DENV-1-4 Real Time RT-PCRCAGGCTATGGCACYGTCACGAT 4 D2-R CDC DENV-1-4 Real Time RT-PCRCCATYTGCAGCARCACCATCTC 5 D2-Probe CDC DENV-1-4 Real Time RT-PCRCTCYCCRAGAACGGGCCTCGACTTCAA 6 D3-F CDC DENV-1-4 Real Time RT-PCRGGACTRGACACACGCACCCA 7 D3-R CDC DENV-1-4 Real Time RT-PCRCATGTCTCTACCTTCTCGACTTGYCT 8 D3-Probe CDC DENV-1-4 Real Time RT-PCRACCTGGATGTCGGCTGAAGGAGCTTG 9 D4-F CDC DENV-1-4 Real Time RT-PCRTTGTCCTAATGATGCTRGTCG 10 D4-R CDC DENV-1-4 Real Time RT-PCRTCCACCYGAGACTCCTTCCA 11 D4-Probe CDC DENV-1-4 Real Time RT-PCRTYCCTACYCCTACGCATCGCATTCCG 12

Processes optionally involve a real-time PCR assay (RT-PCR), optionally,a real-time quantitative PCR assay. In some embodiments, the PCR assayis a TaqMan assay (Holland et al., PNAS 88(16):7276 (1991)). It isappreciated that the processes are amenable to performance on otherRT-PCR systems and protocols that use alternative reagentsillustratively including, but not limited to Molecular Beacons probes,Scorpion probes, multiple reporters for multiplex PCR, combinationsthereof, or other DNA detection systems.

The assays are performed on an instrument designed to perform suchassays, for example those available from Applied Biosystems (FosterCity, Calif.). In some embodiments, a real-time quantitative PCR assayis used to detect the presence of DENV, natural or artificial variants,analogs, or derivatives thereof, in a sample by subjecting the DENVnucleic acid from the sample to PCR reactions using specific primers,and detecting the amplified product using a probe. In some embodiments,the probe is a TaqMan probe which consists of an oligonucleotide with a5′-reporter dye and a 3′-quencher dye.

A fluorescent reporter dye, such as FAM dye (illustratively6-carboxyfluorescein), is covalently linked, optionally to the 5′ end ofthe oligonucleotide probe. Other dyes illustratively include TAMRA,AlexaFluor dyes such as AlexaFluor 495 or 590, Cascade Blue, MarinaBlue, Pacific Blue, Oregon Green, Rhodamine, Fluorescein, TET, HEX, Cy5,Cy3, and Tetramethylrhodamine. A reporter is optionally quenched by adye at the 3′ end or other non-fluorescent quencher. Quenching moleculesare optionally suitably matched to the fluorescence maximum of the dye.Any suitable fluorescent probe for use in RT-PCR detection systems isillustratively operable in the instant invention. Similarly, anyquenching molecule for use in RT-PCR systems is illustratively operable.In some embodiments, a 6-carboxyfluorescein reporter dye is present atthe 5′-end and matched to BLACK HOLE QUENCHER (BHQ1, BiosearchTechnologies, Inc., Novato, Calif.) The fluorescence signals from thesereactions are captured at the end of extension steps as PCR product isgenerated over a range of the thermal cycles, thereby allowing thequantitative determination of the bacterial load in the sample based onan amplification plot.

The DENV nucleic acid sequences are optionally reverse transcribedand/or amplified before or simultaneous with being detected. The term“amplified” defines the process of making multiple copies of the nucleicacid from a single or lower copy number of nucleic acid sequencemolecule. The amplification of nucleic acid sequences is carried out invitro by biochemical processes known to those of skill in the art,illustratively by PCR techniques. The amplification agent may be anycompound or system that will function to accomplish the synthesis ofprimer extension products, including enzymes. Suitable enzymes for thispurpose include, for example, E. coli DNA polymerase I, Taq polymerase,Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, AmpliTaqGold DNA Polymerase from Applied Biosystems, other available DNApolymerases, reverse transcriptase (preferably iScript RNase H+ reversetranscriptase), ligase, and other enzymes, including heat-stable enzymes(i.e., those enzymes that perform primer extension after being subjectedto temperatures sufficiently elevated to cause denaturation). In someembodiments, the enzyme is hot-start iTaq DNA polymerase from Bio-rad(Hercules, Calif.). Suitable enzymes will facilitate combination of thenucleotides in the proper manner to form the primer extension productsthat are complementary to each mutant nucleotide strand. Generally, thesynthesis is initiated at the 3′-end of each primer and proceed in the5′-direction along the template strand, until synthesis terminates,producing molecules of different lengths. There may be amplificationagents, however, that initiate synthesis at the 5′-end and proceed inthe other direction, using the same or similar processes as describedherein. In some examples, the Superscript II platinum One-Step RT-PCRsystem with the PLATINUM Taq DNA polymerase (Invitrogen Corp., Carlsbad,Calif.) is used. In any event, the processes of the invention are not tobe limited to the embodiments of amplification described herein.

One process of in vitro amplification, which optionally is usedaccording to this invention, is the polymerase chain reaction (PCR)described in U.S. Pat. Nos. 4,683,202 and 4,683,195. The term“polymerase chain reaction” refers to a process for amplifying a DNAbase sequence using a heat-stable DNA polymerase and two oligonucleotideprimers, one complementary to the (+)-strand at one end of the sequenceto be amplified and the other complementary to the (−)-strand at theother end. Because the newly synthesized DNA strands can subsequentlyserve as additional templates for the same primer sequences, successiverounds of primer annealing, strand elongation, and dissociation producerapid and highly specific amplification of the desired sequence. Manypolymerase chain processes are known to those of skill in the art andmay be used in the process of the invention. For example, DNA issubjected to 30 to 35 cycles of amplification in a thermocycler asfollows: 2 minutes at 50° C., 10 minutes at 95° C., and then 50×(15seconds at 95° C. plus 1 minute at 60° C.).

The primers for use in amplifying the mRNA or genomic DNA of DENV may beprepared using any suitable process, such as conventionalphosphotriester and phosphodiester processes or automated embodimentsthereof so long as the primers are capable of hybridizing to the nucleicacid sequences of interest. One process for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066. The exact length of primer will depend on many factors,including temperature, buffer, and nucleotide composition. The primermust prime the synthesis of extension products in the presence of theinducing agent for amplification.

Primers used according to the process of the invention are complementaryto each strand of nucleotide sequence to be amplified. The term“complementary” means that the primers hybridize with their respectivestrands under conditions, which allow the agent for polymerization tofunction, such as stringent hybridization conditions. In other words,the primers that are complementary to the flanking sequences hybridizewith the flanking sequences and permit amplification of the nucleotidesequence. Optionally, the 3′ terminus of the primer that is extended isperfectly (100%) base paired with the complementary flanking strand.Probes optionally possess nucleotide sequences complementary to one ormore strands of DENV. Optionally, primers contain the nucleotidesequences of SEQ ID NOs: 1 and 2. It is appreciated that the complementsof SEQ ID NOs: 1 and 2 are similarly suitable for use in the instantinventions. It is further appreciated that oligonucleotide sequencesthat hybridize with SEQ ID NOs 1 or 2 are also similarly suitable.Finally, multiple positions are available for hybridization on DENV andwill be also suitable hybridization with a probe when used with theproper forward and reverse primers.

Those of ordinary skill in the art will know of various amplificationprocesses that can also be utilized to increase the copy number oftarget DENV nucleic acid sequence. The nucleic acid sequences detectedin the process of the invention are optionally further evaluated,detected, cloned, sequenced, and the like, either in solution or afterbinding to a solid support, by any process usually applied to thedetection of a specific nucleic acid sequence such as another polymerasechain reaction, oligomer restriction (Saiki et al., BioTechnology 3:10081012 (1985)), allele-specific oligonucleotide (ASO) probe analysis(Conner et al., PNAS 80: 278 (1983)), oligonucleotide ligation assays(OLAs) (Landegren et al., Science 241:1077 (1988)), RNase ProtectionAssay, among others. Molecular techniques for DNA analysis have beenreviewed (Landegren et al, Science 242:229 237 (1988)). Following DNAamplification, the reaction product may be detected by Southern blotanalysis, with or without using radioactive probes. In such a process,for example, a small sample of DNA containing the nucleic acid sequenceobtained from the tissue or subject is amplified, and analyzed via aSouthern blotting technique. The use of non-radioactive probes or labelsis facilitated by the high level of the amplified signal. In someembodiments of the invention, one nucleoside triphosphate isradioactively labeled, thereby allowing direct visualization of theamplification product by autoradiography. In some embodiments,amplification primers are fluorescently labeled and run through anelectrophoresis system. Visualization of amplified products is by lightdetection followed by computer assisted graphic display, without aradioactive signal.

Other methods of detection amplified oligonucleotide illustrativelyinclude gel electrophoresis, mass spectrometry, liquid chromatography,fluorescence, luminescence, gel mobility shift assay, fluorescenceresonance energy transfer, nucleotide sequencing, enzyme-linkedimmunoadsorbent assay, affinity chromatography, other chromatographymethods, immunoenzymatic methods (Ortiz, A and Ritter, E, Nucleic AcidsRes., 1996; 24:3280-3281), streptavidin-conjugated enzymes, DNA branchmigration (Lishanski, A, et al., Nucleic Acids Res., 2000; 28(9):e42),enzyme digestion (U.S. Pat. No. 5,580,730), colorimetric methods (Lee,K., Biotechnology Letters, 2003; 25:1739-1742), or combinations thereof.A detection signal is produced that is related to the detection methodemployed, be it RT-PCR or other detection method. A test sampleoptionally produces a first detection signal upon amplification of atarget. A control sample optionally produces a second detection signalupon amplification of a control molecule.

The term “labeled” with regard to the probe is intended to encompassdirect labeling of the probe by coupling (i.e., physically linking) adetectable substance to the probe, as well as indirect labeling of theprobe by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a probe using afluorescently labeled antibody and end-labeling or centrally labeling ofa DNA probe with biotin such that it can be detected with fluorescentlylabeled streptavidin. The detection methods can be used to detect DNA,RNA, genomic nucleic acid, or amplification products thereof, in asample in vitro as well as in vivo. For example, in vitro techniques fordetection of nucleic acid include northern hybridizations, in situhybridizations, reverse transcription-PCR, real-time-PCR, and DNaseprotection. In vivo techniques for detection of DENV include introducinginto a subject organism a labeled antibody directed against apolypeptide component or directed against a particular nucleic acidsequence of DENV. For example, the antibody can be labeled with aradioactive marker whose presence and location in the subject organismcan be detected by standard imaging techniques, includingautoradiography.

The size of the primers used to amplify a portion of the nucleic acidsequence of DENV is at least 5, and often 10, 15, 20, 25, 30 or morenucleotides in length, optionally any value or range between 5 and 30nucleotides in length. Optionally, the GC ratio is above 30%, 35%, 40%.45%. 50%, 55%, or 60% so as to prevent hair-pin structure on the primer.The amplicon is optionally of sufficient length to be detected bystandard molecular biology methodologies. The forward primer isoptionally shorter than the reverse primer or vice versa. Techniques formodifying the T_(m) of either primer are operable herein. Anillustrative forward primer or reverse primer contains LNA-dA or LNA-dT(Glen Research Corporation) so as to match T_(m) with a correspondingalternate primer.

A pair of forward and reverse primers optionally hybridize to a targetthat represents the same gene or product of the same gene within one ormore DENV. Illustratively, SEQ ID NOs: 1 and 2 each are directed topositions in the NS5 gene. SEQ ID NOs: 4 and 5 are each directed topositions in the E gene. SEQ ID NOs: 7 and 8 are each directed topositions in the prM gene. SEQ ID NOs: 10 and 11 are each directed topositions in the prM gene. In some embodiments, the amplificationproduct produced by a particular primer pair is unique to that DENVserotype. Illustratively, some embodiments involve a process fordetecting the presence or absence multiple DENV serotypes wherein eachprimer pair recognizes a unique portion of the same gene or differentgenes within DENV.

An inventive process uses a polymerization reaction which employs anucleic acid polymerizing enzyme, illustratively a DNA polymerase, RNApolymerase, reverse transcriptase, or mixtures thereof. It is furtherappreciated that accessory proteins or molecules are present to form thereplication machinery. A polymerizing enzyme is optionally athermostable polymerase or thermodegradable polymerase. Use ofthermostable polymerases is well known in the art such as Taq polymeraseavailable from Invitrogen Corporation, Carlsbad, Calif. Thermostablepolymerases allow a polymerization reaction to be initiated or shut downby changing the temperature other condition in the reaction mixturewithout destroying activity of the polymerase.

Accuracy of the base pairing of DNA sequence amplification is providedby the specificity of the enzyme. Error rates for Taq polymerase tend tobe false base incorporation of 10⁻⁵ or less. (Johnson, Annual Reviews ofBiochemistry, 1993: 62:685-713; Kunkel, Journal of Biological Chemistry,1992; 267:18251-18254). Specific examples of thermostable polymerasesillustratively include those isolated from Thermus aquaticus, Thermusthermophilus, Pyrococcus woesei, Pyrococcus furiosus, Thermococcuslitoralis and Thermotoga maritima. Thermodegradable polymerasesillustratively include E. coli DNA polymerase, the Klenow fragment of E.coli DNA polymerase, T4 DNA polymerase, T7 DNA polymerase and otherexamples known in the art. It is recognized in the art that otherpolymerizing enzymes are similarly suitable illustratively including E.coli, T7, T3, SP6 RNA polymerases and AMV, M-MLV, and HIV reversetranscriptases.

The polymerases are optionally bound to the primer. When the DENVsequence is a single-stranded RNA molecule due to heat denaturing, thepolymerase is bound at the primed end of the single-stranded nucleicacid at an origin of replication. A binding site for a suitablepolymerase is optionally created by an accessory protein or by anyprimed single-stranded nucleic acid.

In some embodiments, detection of PCR products is achieved by massspectrometry. Mass spectrometry has several advantages over real-timePCR systems in that it can be used to simultaneously detect the presenceof DENV and decipher mutations in target nucleic acid sequences allowingidentification and monitoring of emerging strains. Further, massspectrometers are prevalent in the clinical laboratory. Similar tofluorescence based detection systems, mass spectrometry is capable ofsimultaneously detecting multiple amplification products for amultiplexed and controlled approach to accurately quantifying componentsof biological or environmental samples.

Multiple mass spectrometry platforms are suitable for use in theinvention illustratively including matrix assisted laser desorptionionization time of flight mass spectrometry (MALDI), electrospray massspectrometry, electrospray ionization-Fourier transform ion cyclotronresonance mass spectrometry (ESI-FTICR), multi-stage mass spectrometryfragmentation analysis (MS/MS), mass spectrometry coupled with liquidchromatography such as high performance liquid chromatography massspectrometry (HPLC) and ultra performance liquid chromatography isotopedilution tandem mass spectrometry (UPLC-ID/MS/MS), and variationsthereof.

It is appreciated that numerous other detection processes are similarlysuitable for measuring an amplification product by detecting a detectionsignal. Illustrative examples include, but are not limited to, liquidchromatography, mass spectrometry, liquid chromatography/massspectrometry, static fluorescence, dynamic fluorescence, highperformance liquid chromatography, ultra-high performance liquidchromatography, enzyme-linked immunoadsorbent assay, real-time PCR(RT-PCR), gel electrophoresis, or combinations thereof.

Optionally, PCR amplification products are generated using complementaryforward and reverse oligonucleotide primers. In a non-limiting example,DENV-1 genetic sequences or fragments thereof are amplified by theprimer pair SEQ ID NOs: 1 and 2. The resulting amplification product iseither directly detected such as by a probe, or is subsequentlyprocessed and prepared for detection by processes known in the art. Itis appreciated that the complements of SEQ ID NOs: 1 and 2 are similarlysuitable for use in the invention. It is further appreciated thatoligonucleotide sequences that hybridize with SEQ ID NOs: 1 or 2 arealso similarly suitable. Finally, multiple positions are available forhybridization on the DENV genome, or other and will be also suitablehybridization with forward and reverse primers that may or may not beused with a probe for PCR.

A primer pair is optionally SEQ ID NOs: 1 and 2, SEQ ID NOs: 4 and 5,SEQ ID NOs: 7 and 8, or SEQ ID NOs: 10 and 11.

In some embodiments, a multiplex assay or set of singleplex assays areperformed. Optionally, an assay detects the presence or absence ofDENV-1. Optionally, an assay detects the presence or absence of DENV-2.Optionally, an assay detects the presence or absence of DENV-3.Optionally, an assay detects the presence or absence of DENV-4.Optionally, an assay detects the presence or absence of DENV-1 andDENV-2. Optionally, an assay detects the presence or absence of DENV-1and DENV-3. Optionally, an assay detects the presence or absence ofDENV-1 and DENV-4. Optionally, an assay detects the presence or absenceof DENV-2 and DENV-3. Optionally, an assay detects the presence orabsence of DENV-2 and DENV-4. Optionally, an assay detects the presenceor absence of DENV-3 and DENV-4. Optionally, an assay detects thepresence or absence of DENV-1, DENV-2 and DENV-3. Optionally, an assaydetects the presence or absence of DENV-1, DENV-2 and DENV-4.Optionally, an assay detects the presence or absence of DENV-2, DENV-3and DENV-4. Optionally, an assay detects the presence or absence ofDENV-1, DENV-3 and DENV-4. Optionally, an assay detects the presence orabsence of DENV-1, DENV-2, DENV-3 and DENV-4. Optionally an assaydetects the presence or absence of 1, 2, 3, or 4 serotypes selected fromDENV-1, DENV-2, DENV-3 and DENV-4.

Optionally, multiple amplification products are simultaneously producedin a multiplex PCR reaction that are then available for simultaneousdetection and quantification. Thus, multiple detection signals areinherently produced or emitted that are separately and uniquely detectedin one or more detection systems. In some embodiments, the primer setsand probes of Table 1 are all simultaneously used in single reactionchamber in a single multiplex reaction. Optionally, the primer pairs andrelated probes of Table 1 are used in a plurality of singleplexreactions with each tube possessing sufficient primers and probes torecognize one or more DENV serotypes. One or more singleplex reactionsare optionally performed simultaneously or sequentially. Multipledetection signals produced by multiple probes are optionally produced inparallel. Optionally, a single biological sample is subjected toanalysis for the simultaneous or sequential detection of DENV geneticsequences. It is appreciated that three or more independent oroverlapping sequences are simultaneously or sequentially measured in theinventive processes. Oligonucleotide matched primers (illustratively SEQID NOs: 1 and 2) are simultaneously or sequentially added and thebiological sample, or a portion thereof, is subjected to properthermocycling reaction parameters. For detection by mass spectrometry, asingle sample of the amplification products from each gene aresimultaneously analyzed allowing for rapid and accurate determination ofthe presence of DENV. Optionally, analysis by real-time PCR is employedcapitalizing on multiple probes with unique fluorescent signatures.Thus, each gene is detected without interference by other amplificationproducts. This multi-target approach increases confidence inquantification and provides for additional internal control.

In some embodiments, the processes further involve optionally obtaininga control sample from a control subject, contacting a control sample,optionally from said subject, with a compound or agent capable ofdetecting the presence of DENV nucleic acid in the sample, and comparingthe presence or absence of RNA or DNA in the control sample with thepresence of RNA or DNA in the test sample. A control sample isoptionally a portion of a test sample processed in parallel with thetest sample. A control sample is optionally a purified, isolated, orotherwise processed nucleic acid sequence of known concentrationoptionally including at least a portion of DENV sequence, where thenucleic acid sequence or portion thereof will hybridize under stringentconditions with a forward primer, a reverse primer, and, optionally, aprobe. A control sample is used to produce a complementary amplificationproduct produced either simultaneously with, or sequentially to thefirst amplification product produced from a target. The complementaryamplification product is optionally detected by detecting a seconddetection signal by the same of a different method than that used todetect the first amplification product. Illustratively, a secondamplification product is detected using a second probe of the same or ofa different sequence than that use to detect the first amplificationproduct. A second probe optionally has one or more labels that are thesame or different than that of a first probe, when present.Illustratively, a control sample is subjected to the identicalamplification conditions in the same or other parallel analysis, such ason the same instrument, as the test sample. If the test sample and thecontrol sample are processed in different reaction chambers, the sameprobes with the same labels may be used.

Some embodiments include using a nucleic acid calibrator to produce asignal from a known quantity of sample molecule. A nucleic acidcalibrator is optionally identical to or different from a targetmolecule. Amplification of a nucleic acid calibrator optionally producesa third detection signal, the presence of, intensity of, or size of isoptionally compared to a first detection signal to quantify the amountof target, or amplification product in the test sample. Optionally, aplurality of nucleic acid calibrators are used. A plurality of nucleicacid calibrators are optionally of differing concentrations such asthose suitable to produce a standard curve. The detection signal fromthe test sample is optionally compared to the standard curve to quantifythe amount of amplification product or target in the test sample. Anucleic acid calibrator optionally includes a known amount of DENVnucleic acid sequence, or a portion of a DENV nucleic acid sequence.

Also provided are one or more kits for detecting or diagnosing DENVinfection that contains reagents for the amplification, or directdetection of DENV or portions thereof in a sample. An exemplary kitoptionally includes a forward and reverse primer pair, and a probe. Kitsinclude any primer, probe, or set of primers or probes defined herein,analogues there, variants thereof, or derivatives thereof. It is furtherappreciated that a kit optionally includes ancillary reagents such asbuffers, solvents, thermostable polymerases, nucleotides, and otherreagents necessary and recognized in the art for amplification anddetection of DENV in a sample.

A kit for detection of DENV infection in a subject optionally containsreagents for PCR based detection of DENV genetic sequences, eitherstructural or non-structural, and optionally for detection of antibodiesdirected to DENV proteins. The components of the kits are any of thereagents described above or other necessary and non-necessary reagentsknown in the art for solubilization, detection, washing, storage, orother need for in a diagnostic assay kit. Suitable antibodies are knownin the art.

The invention also encompasses kits for detecting the presence of DENVnucleic acids in a test sample. The kit, for example, includes a labeledcompound or agent capable of detecting a nucleic acid molecule in a testsample and, in certain embodiments, for determining the quantity of DENVin the sample.

For oligonucleotide-based kits, the kit includes, for example: (1) anoligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence of DENV and/or (2) one or a pairof primers (one forward and one reverse) useful for amplifying a nucleicacid molecule containing at least a portion the DENV sequence. The kitcan also include, e.g., a buffering agent, a preservative, or a proteinstabilizing agent. The kit can also include components necessary fordetecting the detectable agent (e.g., an enzyme or a substrate). The kitcan also contain a control sample or a series of control samples whichis assayed and compared to the test sample contained. Each component ofthe kit is usually enclosed within an individual container and all ofthe various containers are usually enclosed within a single packagealong with instructions for use.

Also provided are a library of nucleic acid primers and probes suitablefor use in diagnosis of DENV infection, detection of the presence orabsence of DENV in a sample, or for combination with other kits suitablefor such purposes. A library optionally includes a pair of primers and aprobe for detection of DENV-1. A forward primer is optionally SEQ IDNO: 1. A reverse primer is optionally SEQ ID NO: 2. A probe isoptionally SEQ ID NO: 3. A library or kit optionally includes a pair ofprimers and a probe (s) for detection of DENV-2. A forward primer isoptionally SEQ ID NO: 4. A reverse primer is optionally SEQ ID NO: 5. Aprobe is optionally SEQ ID NO: 6. A library optionally includes a pairof primers and a probe for detection of DENV-3. A forward primer isoptionally SEQ ID NO: 7. A reverse primer is optionally SEQ ID NO: 8. Aprobe is optionally SEQ ID NO: 9. A library optionally includes a pairof primers and a probe for detection of DENV-4. A forward primer isoptionally SEQ ID NO: 10. A reverse primer is optionally SEQ ID NO: 11.A probe is optionally SEQ ID NO: 12. A pair (or more) of forwardprimers, reverse primers, probes, or both with each sequence of a primeror probe of the degeneracy represented in the library or kit. Librariesor kits are provided that include a sufficient number of primers orprobes to represent each degenerate nucleic acid at the indicatedpositions in Table 1. A kit or a library optionally includes primers orprobes for DENV-1, DENV-2, DENV-3, and DENV-4, or any subgroup thereof.

The processes are amenable to use for diagnosis of DENV infection orsimple detection of the presence of DENV in a subject, such as insects,and any other organism capable of infection or transfection by or withDENV.

To increase confidence and to serve as an internal or external control,a purified solution containing DENV is optionally used as a sample.Optionally, by amplification of a single sample with known quantities ofDENV or of a set of samples representing a titration of DENV, the levelof DENV in the unknown biological sample is determined, optionally as acontrol. Optionally, the purified and quantified DENV solution isanalyzed in parallel with the unknown biological sample to reduce interassay error or to serve as a standard curve for quantitation of unknownDENV in the test sample. Using purified and quantified DENV solutionprovides for a similar complete genetic base RNA strand foramplification.

In some embodiments, a subgenomic fragment is cloned into a plasmid foramplification, purification, and use as a quantitative comparator ornucleic acid calibrator. In a non-limiting example, a RNA fragment ofDENV is optionally amplified from a positive serum sample using primersbracketing the RT-PCR target regions in DENV. The known concentration ofthe subgenomic fragment is used to create a standard curve forquantitative determinations and to access amplification efficiency.

Various aspects of the present invention are illustrated by thefollowing non-limiting examples. The examples are for illustrativepurposes and are not a limitation on any practice of the presentinvention. It will be understood that variations and modifications canbe made without departing from the spirit and scope of the invention.While the examples are generally directed to samples derived from ahuman, a person having ordinary skill in the art recognizes that similartechniques and other techniques known in the art readily translate theexamples to other organisms. Reagents illustrated herein are commonlycross reactive between mammalian species or alternative reagents withsimilar properties are commercially available, and a person of ordinaryskill in the art readily understands where such reagents may beobtained.

EXAMPLES Example 1 RT-PCR Assay Design

Serotype-specific DENV primers and fluorogenic probes are designed byusing a newly generated consensus sequence based on multiple new entriesinto the database of DENV genetic sequences. The DENV consensus sequenceis entered into Primer Express 3.0 (Applied Biosystems). Each of theprimers and probes is used to generate families of primers and probeswith various mutations or degeneracies introduced at selected regions ofthe primers or probes. Every primer and probe is aligned to thecorresponding serotype alignment and the frequency of every base in theoligonucleotide is determined. Mismatches between prior primers/probesand the sequence alignments are attended to as follows: degeneracy isintroduced in a given nucleotide position when mismatch is 40% orgreater between 2 or more strains; a base is replaced by another fixedbase when mismatch is 90% or greater between 2 or more strains in agiven position. International Union of Pure and Applied Chemistry(IUPAC) codes are used for all degenerate bases. These new primer andprobe sequences identified take into consideration the availablesequences of contemporary DENV lineages. Primers and probes are analyzedfor homology to other known sequences using the Basic Local AlignmentSearch Tool (BLAST). Altschul S F, et al., J Mol Biol, 1990; 215:403-410. These primers and probes, which are not identified by thePrimer Express tool, are studied for possible amplification of DENVsequences. The identified primers and probes are compared to referencesequences as in Table 2 with the bold nucleotide representing adegenerate or non-wild type substitution.

TABLE 2 Oligonucleotide D1-F Reference (24) CAAAAGGAAGTCGTGCAATA(SEQ ID NO: 13) CDC DENV-1-4 Real Time RT- CAAAAGGAAGTCGYGCAATA PCR(SEQ ID NO: 1) D1-R Reference (24) CTGAGTGAATTCTCTCTACTGAAC(SEQ ID NO: 14) CDC DENV-1-4 Real Time RT- CTGAGTGAATTCTCTCTGCTRAAC PCR(SEQ ID NO: 2) D1-Probe Reference (24) CATGTGGTTGGGAGCACGC(SEQ ID NO: 15) CDC DENV-1-4 Real Time RT- CATGTGGYTGGGAGCRCGC PCR(SEQ ID NO: 3) D2-F Reference (24) CAGGTTATGGCACTGTCACGAT(SEQ ID NO: 16) CDC DENV-1-4 Real Time RT- CAGGCTATGGCACYGTCACGAT PCR(SEQ ID NO: 4) D2-R Reference (24) CCATCTGCAGCAACACCATCTC(SEQ ID NO: 17) CDC DENV-1-4 Real Time RT- CCATYTGCAGCARCACCATCTC PCR(SEQ ID NO: 5) D2-Probe Reference (24) CTCTCCGAGAACAGGCCTCGACTTCAA(SEQ ID NO: 18) CDC DENV-1-4 Real Time RT- CTCYCCRAGAACGGGCCTCGACTTCAAPCR (SEQ ID NO: 6) D3-F Reference (24) GGACTGGACACACGCACTCA(SEQ ID NO: 19) CDC DENV-1-4 Real Time RT- GGACTRGACACACGCACCCA PCR(SEQ ID NO: 7) D3-R Reference (24) CATGTCTCTACCTTCTCGACTTGTCT(SEQ ID NO: 20) CDC DENV-1-4 Real Time RT- CATGTCTCTACCTTCTCGACTTGYCTPCR (SEQ ID NO: 8) D3-Probe Reference (24) ACCTGGATGTCGGCTGAAGGAGCTTG(SEQ ID NO: 21) CDC DENV-1-4 Real Time RT- ACCTGGATGTCGGCTGAAGGAGCTTGPCR (SEQ ID NO: 9) D4-F Reference (24) TTGTCCTAATGATGCTGGTCG(SEQ ID NO: 22) CDC DENV-1-4 Real Time RT- TTGTCCTAATGATGCTRGTCG PCR(SEQ ID NO: 10) D4-R Reference (24) TCCACCTGAGACTCCTTCCA (SEQ ID NO: 23)CDC DENV-1-4 Real Time RT- TCCACCYGAGACTCCTTCCA PCR (SEQ ID NO: 11)D4-Probe Reference (24) TTCCTACTCCTACGCATCGCATTCCG (SEQ ID NO: 24)CDC DENV-1-4 Real Time RT- TYCCTACYCCTACGCATCGCATTCCG PCR(SEQ ID NO: 12)

The DENV-1 probe is labeled at the 5′ end with the 6-carboxyfluorescein(FAM) reporter dye and at the 3′ end with black hole quencher 1 (BHQ-1)fluorophore; the DENV-2 probe is labeled with HEX and BHQ-1; the DEN-3probe is labeled with Texas red (TR) and BHQ-2; and the DEN-4 probe islabeled with Cy5 and BHQ-3. Genetic sequences from non DENV flavivirusesare used as controls.

RT-PCR is performed as follows: In singleplex reaction mixtures, 5 μl ofRNA are combined with 25 pmol of each primer and 4.5 pmol of the probein a 25 μl total volume reaction using Superscript III One Step RT-PCR(Invitrogen, Carlsbad, Calif.). Each reaction mixture contains a singleDENV serotype primer pair (with degenerate sequence) and probe (withdegenerate sequence); therefore, in singleplex assays, four separatereactions are carried out for each RNA sample. In fourplex reactionmixtures (e.g. multiplex), 5 μl of RNA are combined with 25 pmol of eachprimer of DENV-1 and DENV-3, 12.5 pmol of each primer of DENV-2 andDENV-4 (each), and 4.5 pmol of each respective probe (primers and probesused are listed in Table 2) in a 25 μl volume total reaction mixture.Reverse transcription of 30 min at 50° C. is followed by 45 cycles ofamplification in an ABI 7500 Dx (Applied Biosystems, Foster City,Calif.) according to Invitrogen Superscript instructions for real-timeRT-PCR conditions and using a 60° C. annealing temperature.

Identification of Limit of Detection (LOD)

The CDC DENV-1-4 assay limit of detection (LoD) is determined using apanel of quantified (pfu/mL) stocks of laboratory strain infectiousDENV-1, -2, -3 and -4 diluted in human serum or plasma with eight (8)1:10 dilutions; 5 replicas per dilution. Viral RNA from every replicasample is extracted and tested using the protocol above. The assay isrun with a quantitative component in order to compare the differentversions of the assay with improved accuracy. The LoD of the assay isdefined as the last dilution in which virus is detected in 100% of thereplicas. Virus detection, measured in genome copy equivalents per mL ofsample (GCE/mL) is compared between virus dilutions in human serum andhuman plasma and the results illustrated in FIG. 1A (singleplex) and 1B(multiplex). Equivalent detection of DENV serotypes is achieved in both,singleplex and multiplex formats of the assay diluted in serum orplasma. The LoD is measured at approximately 1×10⁴ GCE/mL in bothformats, which corresponds to titers of approximately 1×10³ pfu/mL.

Broad Recognition of DENV Strains

The assay is further tested on 29 DENV-1-4 cultured and quantifiedclinical isolates obtained from international locations in order toconfirm that the chosen assay conditions above will broadly recognizeDENV strains from a broad geographical distribution of sources.Quantified stocks are serially diluted in human serum at 1:10 dilutionsdown to 1×10² pfu/mL in triplicate. A similar LoD is determined in allcultured clinical isolates as is illustrated in Table 3 depicting theyear of the infection's source, the location the sample was taken, andthe genotype of the DENV strain.

TABLE 3 10³ pfu/ml 10² pfu/ml Serotype Year Country Genotype Rate PosRate Pos DENV-1 2003 Brazil African/ 3/3 0/3 American DENV-1 2007 MexicoAfrican/ 3/3 0/3 American DENV-1 2007 Venezuela African/ 3/3 0/3American DENV-1 1994 Sri Lanka South Pacific 3/3 0/3 DENV-1 2004Philippines South Pacific 3/3 0/3 DENV-1 2004 Thailand Asian 3/3 0/3DENV-1 2006 Thailand Asian 3/3 0/3 DENV-2 2006 Brazil SE Asian/ 3/3 0/3American DENV-2 2007 Colombia SE Asian/ 3/3 0/3 American DENV-2 1980Ivory Coast Sylvatic 3/3 2/3 DENV-2 1988 Viet Nam Asian II 3/3 1/3DENV-2 2006 Thailand Asian II 3/3 0/3 DENV-2 2003 Dominican SE Asian/3/3 1/3 R. American DENV-2 2003 Costa Rica SE Asian/ 3/3 0/3 AmericanDENV-2 1996 Peru American 3/3 1/3 DENV-2 1982 Burkina Faso Cosmopolitan3/3 1/3 DENV-2 2006 India Cosmopolitan 3/3 0/3 DENV-3 2006 Puerto RicoIndian Subcont. 3/3 0/3 DENV-3 2003 Brazil Indian Subcont. 3/3 1/3DENV-3 1995 Samoa South Pacific 3/3 0/3 DENV-3 2006 Thailand Thailand3/3 0/3 DENV-3 2000 Ecuador Indian Subcont. 3/3 1/3 DENV-3 1991 CookIsland South Pacific 3/3 0/3 DENV-4 2006 Colombia Indonesian 3/3 0/3DENV-4 2006 Mexico Indonesian 3/3 0/3 DENV-4 1992 Sri Lanka SE Asian 3/31/3 DENV-4 2006 Thailand SE Asian 3/3 0/3 DENV-4 1994 St. CroixIndonesian 3/3 0/3 DENV-4 1999 Ecuador Indonesian 3/3 1/3 DENV-4 1995Micronesia SE Asian 3/3 0/3Cross Reactivity

The assay above is further evaluated for analytical specificity bytesting with nucleic acids extracted from 12 organisms representingcommon pathogens present in the blood of patients with febrile illness.The above assay is performed on all 12 samples in triplicate. Allnegative samples test negative for DENV (Table 4). Only the samplesspiked with DENV test positive indicating that the assay presents nocross-reactivity with any pathogen tested at clinically relevantconcentrations.

TABLE 4 DENV RT-PCR Pathogen Sample type Concentration Rate positiveVirus pfu/ml DENV-1 spiked serum   1 × 10⁶ 3/3 DENV-2 spiked serum   1 ×10⁶ 3/3 DENV-3 spiked serum   1 × 10⁶ 3/3 DENV-4 spiked serum   1 × 10⁶3/3 WNV spiked serum 6.9 × 10⁷ 0/3 YFV spiked serum 3.7 × 10⁶ 0/3 SLEVspiked serum 3.7 × 10⁶ 0/3 CHIKV spiked serum 4.0 × 10⁶ 0/3 HCV clinicalserum unknown 0/3 HAV clinical serum unknown 0/3 HSV-1 spiked serum 1.0× 10⁵ 0/3 HSV-2 spiked serum 1.0 × 10⁵ 0/3 CMV spiked serum 1.0 × 10⁵0/3 VZV spiked serum 1.0 × 10⁵ 0/3 Bacteria bacteria/ml Leptospirospiked serum 2.5 × 10⁵ 0/3 Borrelia spiked serum 1.0 × 10⁶ 0/3burgdorferiInterference with Potentially Contaminating Sample Materials

The assay above is evaluated in the presence of normal human serum (NHS)or in NHS containing bilirubin, cholesterol, hemoglobin, triglycerides,or genomic DNA. Every interference study is performed in the presence ofcultured and quantified (pfu/mL) stocks of infectious laboratory strainDENV-1-4 diluted to concentrations 1:10 above the LoD dilution (1×10⁴pfu/ml), equal to the LoD dilution (1×10³ pfu/ml), and the 1:10 dilutionbelow the LoD (1×10² pfu/ml). No significant interference in DENVdetection is observed in the presence of any of the potential humanendogenous interfering biomolecules tested.

Carryover or Cross-Contamination Studies

A panel of DENV-1-4 diluted to high-positive (10⁷ pfu/ml) andhigh-negative (5×10² pfu/ml) concentrations are used to determinesensitivity of the above assay to cross contamination. Eighthigh-positive and 8 high-negative replicas are tested in an alternatingseries. Viral RNA from every replica sample is extracted using theQiagen QIAamp® DSP Viral RNA Mini Kit and the assay is performed asabove. All negative samples test negative (32/32) and all DENV positivesamples test positive for DENV (32/32) indicating no observed crosscontamination.

Effect of Sample Freeze/Thaw on Assay Detection Ability

Effects of temperature variation on the above assay performance areevaluated by comparing detection of DENV spiked human serum samplesafter multiple freeze/thaw cycles using the above assay procedure.Moderate and low positive dilutions are prepared in triplicate andfrozen at −80° C. for 24 hours and then subjected to five consecutivefreeze/thaw cycles. Once thawed, every sample is processed accordingly.This validation determined a 100% qualitative agreement between theinitial and post freeze/thaw cycle (1, 2, 3, 4, and 5) detection resultsas illustrated in FIG. 2.

Example 2 Assay for Presence of DENV in Biological Samples from ClinicalSources

The ability of the DENV assay of Example 1 to detect DENV usingextracted RNA from serum obtained from either control subjects or fromsubjects diagnosed as DENV positive by serological assays is evaluated.The RT-PCR assay of Example 1 is used to examine each of the samples forthe presence or absence of DENV. Results are illustrated in Table 5where the degenerate and other non-wild-type substitutions arehighlighted in bold.

TABLE 5 Fre- Amplicon Oligonucleotide quency Position Gene size D1-FReference (24) CAAAAGGAAGTCGTGCAATA 12/16 8936-8955 NS5 112 bp(SEQ ID NO: 13) CDC DENV-1-4 Real CAAAAGGAAGTCGYGCAATA 16/16 Time RT-PCR(SEQ ID NO: 1) D1-R Reference (24) CTGAGTGAATTCTCTCTACTGAAC  3/169023-9047 NS5 112 bp (SEQ ID NO: 14) CDC DENV-1-4 RealCTGAGTGAATTCTCTCTGCTRAAC 16/16 Time RT-PCR (SEQ ID NO: 2) D1-Reference (24) CATGTGGTTGGGAGCACGC  4/16 8961-8979 NS5 112 bp Probe(SEQ ID NO: 15) CDC DENV-1-4 Real CATGTGGYTGGGAGCRCGC 16/16 Time RT-PCR(SEQ ID NO: 3) D2-F Reference (24) CAGGTTATGGCACTGTCACGAT  0/161426-1447 E  78 bp (SEQ ID NO: 16) CDC DENV-1-4 RealCAGGCTATGGCACYGTCACGAT 13/16 Time RT-PCR (SEQ ID NO: 4) D2-RReference (24) CCATCTGCAGCAACACCATCTC  3/16 1482-1504 E  78 bp(SEQ ID NO: 17) CDC DENV-1-4 Real CCATYTGCAGCARCACCATCTC 16/16Time RT-PCR (SEQ ID NO: 5) D2- Reference (24)CTCTCCGAGAACAGGCCTCGACTTCAA 10/16 1454-1480 E  78 bp Probe(SEQ ID NO: 18) CDC DENV-1-4 Real CTCYCCRAGAACGGGCCTCGACTTCAA 14/16Time RT-PCR (SEQ ID NO: 6) D3-F Reference (24) GGACTGGACACACGCACTCA 9/15 701-720 prM  74 bp (SEQ ID NO: 19) CDC DENV-1-4 RealGGACTRGACACACGCACCCA 15/15 Time RT-PCR (SEQ ID NO: 7) D3-RReference (24) CATGTCTCTACCTTCTCGACTTGTCT  5/15 749-775 prM  74 bp(SEQ ID NO: 20) CDC DENV-1-4 Real CATGTCTCTACCTTCTCGACTTGYCT 15/15Time RT-PCR (SEQ ID NO: 8) D3- Reference (24) ACCTGGATGTCGGCTGAAGGAGCTTG14/15 722-747 prM  74 bp Probe (SEQ ID NO: 21) CDC DENV-1-4 RealACCTGGATGTCGGCTGAAGGAGCTTG 14/15 Time RT-PCR (SEQ ID NO: 9) D4-FReference (24) TTGTCCTAATGATGCTGGTCG 4/7 884-904 prM  89 bp(SEQ ID NO: 22) CDC DENV-1-4 Real TTGTCCTAATGATGCTRGTCG 7/7 Time RT-PCR(SEQ ID NO: 10) D4-R Reference (24) TCCACCTGAGACTCCTTCCA 5/7 953-973 prM 89 bp (SEQ ID NO: 23) CDC DENV-1-4 Real TCCACCYGAGACTCCTTCCA 7/7Time RT-PCR (SEQ ID NO: 11) D4- Reference (24)TTCCTACTCCTACGCATCGCATTCCG 4/7 939-965 prM  89 bp Probe (SEQ ID NO: 24)CDC DENV-1-4 Real TYCCTACYCCTACGCATCGCATTCCG 7/7 Time RT-PCR(SEQ ID NO: 12)

Briefly, the primers and probes of SEQ ID NOs: 1-12 show greatsuperiority in their capability to recognize each DENV serotype asillustrated by far superior frequency of recognition of the targetvirus. For the first time, this assay is robust enough to be used in adetection assay whereby a positive result demonstrates diagnosis of DENVinfection in a subject, and the absence of a positive result diagnosesthe absence of DENV infection in the subject.

Example 3 Detection of DENV is Clinical Samples in a Prospective Study

A total of 86 acute serum samples are collected from dengue-suspected,febrile patients (0-5 days of symptoms; AVG age 14.3, 42% F) at 3different public health laboratories (2009-2011). Fifty (50) serumsamples are obtained through the dengue fever passive surveillancesystem administered by the CDC Dengue Branch: 25 of these serum samplesare tested at the CDC Dengue Branch Laboratory and 25 serum samples aredecoded, sent to, and tested at the Puerto Rico Department of Health.Thirty six (36) serum samples are obtained as part of a nationalsurveillance and reference program in Costa Rica. Samples are receivedby the National Laboratory in Costa Rica and decoded before testing. Allthree laboratories follow the same protocol: viral RNA is extractedusing the Qiagen QIAamp® DSP Viral RNA Mini Kit (cat#61904) followingthe manufacturer's protocol. The eluted viral RNA is tested by the assayof Example 1 using the primers and probes of Table 1. To confirmdiagnosis, the DENV envelope gene is sequenced on all 86 samples usingbi-directional Sanger sequencing. Applying sequence data as a referencemethod, the assay achieved a 97.92% positive agreement and 100% negativeagreement (Table 6). The prospective samples are not complemented by asecond, convalescent specimen.

TABLE 6 Multiplex CDC DENV-1-4 Real-Time RT-PCR Assay Comparison ResultsReference Method (Sequencing) Positive Negative Total CDC DENV-1-4 Real-Positive 47 0 47 Time RT-PCR Assay Negative  1* 38 39 Total 48 38 86Value 95% Confidence Interval Positive percent agreement 97.92%89.10-99.63 Negative percent agreement   100% 90.82-100  

Example 4 Detection of DENV is Clinical Samples in a Retrospective Study

The assay of Example 1 is further evaluated using retrospective clinicalsamples obtained from the archived CDC routine dengue surveillancespecimens collected in pairs (2007-2011). Acute samples were collectedduring the first five days of symptoms, and convalescent sample wascollected at least 6 days after the onset of symptoms. These samples aretested with the IgM Capture Enzyme Linked Immunosorbent Assay (CDCMAC-ELISA—validated in-house) in order to establish seroconversion. Atotal of 371 acute samples: 39 dengue-positive international samples, 82dengue-positive samples from the Puerto Rico dengue surveillance system,and an additional 250 dengue-negative samples (no IgM conversion), alsofrom Puerto Rico, are included in the test. The percent (%) agreementbetween the result using the assay of Example 1 and the IgM conversionis calculated for the number of samples that received positive ornegative results and the results presented in Table 7. In addition,bi-directional Sanger sequencing of the DENV E gene is performed tocorroborate CDC DENV-1-4 Real-Time RT-PCR positive detection andserotyping results.

TABLE 7 Multiplex CDC DENV-1-4 Real-Time RT-PCR Assay Comparison ResultsReference Method (IgM Conversion)^(†) Positive Negative Total CDCDENV-1-4 Positive 100*    4*** 104 Real-Time RT- Negative   2** 265 267PCR Assay Total 102 269 371 Value 95% Confidence Interval Positivepercent agreement 98.04% 93.13-99.46 Negative percent agreement 98.51%96.24-99.42

Example 5 Detection of DENV by PCR/LC/MS

The samples of Example 2 are each rescreened using RT-PCR amplificationwith parameters similar to the RT-PCR assay of Example 1. The reactionproducts are subjected to analyses by electrospray ionization massspectrometry substantially as described by Naito, Y, et al., RapidCommunications in Mass Spectrometry, 1995; 9:1484-1486; or Wunschel D S,et al., Rapid Commun Mass Spectral. 1996; 10(1):29-35. Each of thereaction products from the PCR reactions are successfully and rapidlydetected.

Example 6 Detection of DENV by PCR/Gel Electrophoresis

Samples of Example 2 are each rescreened using PCR amplification withparameters similar to the RT-PCR assay of Example 1. The amplifiedreaction products are separated by gel electrophoresis and detected byfluorescent imaging. Each of the primer pairs and probes show detectableamplified DENV.

Methods involving conventional biological techniques are describedherein. Such techniques are generally known in the art and are describedin detail in methodology treatises such as Molecular Cloning: ALaboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates); andShort Protocols in Molecular Biology, ed. Ausubel et al., 52 ed.,Wiley-Interscience, New York, 2002. Immunological methods (e.g.,preparation of antigen-specific antibodies, immunoprecipitation, andimmunoblotting) are described, e.g., in Current Protocols in Immunology,ed. Coligan et al., John Wiley & Sons, New York, 1991; and Methods ofImmunological Analysis, ed. Masseyeff et al., John Wiley & Sons, NewYork, 1992.

Additional protocols such as PCR Protocols can be found in A Guide toMethods and Applications Academic Press, NY.

Various modifications of the present invention, in addition to thoseshown and described herein, will be apparent to those skilled in the artof the above description. Such modifications are also intended to fallwithin the scope of the appended claims.

It is appreciated that all reagents are obtainable by sources known inthe art unless otherwise specified. Methods of nucleotide amplification,cell transfection, and protein expression and purification are similarlywithin the level of skill in the art.

Various modifications of the present invention, in addition to thoseshown and described herein, will be apparent to those skilled in the artof the above description. Such modifications are also intended to fallwithin the scope of the appended claims.

REFERENCE LIST

-   1. Alexander, N., A. Balmaseda, I. C. Coelho, E. Dimaano, T. T.    Hien, N. T. Hung, T. Janisch, A. Kroeger, L. C. Lum, E.    Martinez, J. B. Siqueira, T. T. Thuy, I. Villalobos, E. Villegas, B.    Wills, and W. H. O. s. D. S. G. on behalf of the European    Union. 2011. Multicentre prospective study on dengue classification    in four South-east Asian and three Latin American countries. Trop    Med Int Health.-   2. Ayala, A., A. Rivera, M. Johansson, and J. L. Muñoz-Jordán. 2006.    Travel-Associated Dengue—United States, 2005. MMWR 55:700-702.-   3. Bessoff, K., M. Delorey, W. Sun, and E. Hunsperger. 2008.    Comparison of two commercially available dengue virus (DENV) NS1    capture enzyme-linked immunosorbent assays using a single clinical    sample for diagnosis of acute DENV infection. Clin Vaccine Immunol    15:1513-1518.-   4. Brunkard, J. M., J. L. Robles Lopez, J. Ramirez, E.    Cifuentes, S. J. Rothenberg, E. A. Hunsperger, C. G. Moore, R. M.    Brussolo, N. A. Villarreal, and B. M. Haddad. 2007. Dengue Fever    Seroprevalence and Risk Factors, Texas-Mexico Border, 2004. Emerg    Infect Dis 13:1477-1483.-   5. Callahan, J. D., S. J. Wu, A. Dion-Schultz, B. E. Mangold, L. F.    Peruski, D. M. Watts, K. R. Porter, G. R. Murphy, W.    Suharyono, C. C. King, C. G. Hayes, and J. J. Temenak. 2001.    Development and evaluation of serotype- and group-specific    fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue    virus. J Clin Microbiol 39:4119-4124.-   6. Centers for Disease, C., and Prevention. 2010. Locally acquired    Dengue—Key West, Fla., 2009-2010. MMWR. Morbidity and mortality    weekly report 59:577-581.-   7. Chau, T. N., K. L. Anders, B. Lien le, N. T. Hung, L. T.    Hieu, N. M. Tuan, T. T. Thuy, T. Phuong le, N. T. Tham, M. N.    Lanh, J. J. Farrar, S. S. Whitehead, and C. P. Simmons. 2010.    Clinical and virological features of Dengue in Vietnamese infants.    PLoS Negl Trop Dis 4:e657.-   8. Chien, L. J., T. L. Liao, P. Y. Shu, J. H. Huang, D. J. Gubler,    and G. J. Chang. 2006. Development of real-time reverse    transcriptase PCR assays to detect and serotype dengue viruses. J    Clin Microbiol 44:1295-1304.-   9. Condon, R., G. Taleo, T. Stewart, T. Sweeney, and T.    Kiedrzynski. 2000. Dengue surveillance in the Pacific Islands.    Pacific health dialog 7:122-126.-   10. Descloux, E., V. M. Cao-Lormeau, C. Roche, and X. De    Lamballerie. 2009. Dengue 1 diversity and microevolution, French    Polynesia 2001-2006: connection with epidemiology and clinics. PLoS    Negl Trop Dis 3:e493.-   11. Dussart, P., L. Petit, B. Labeau, L. Bremand, A. Leduc. D.    Moua, S. Matheus, and L. Baril. 2008. Evaluation of Two New    Commercial Tests for the Diagnosis of Acute Dengue Virus Infection    Using NS1 Antigen Detection in Human Serum. PLoS Negl Trop Dis    2:e280.-   12. Effler, P. V., L. Pang, P. Kitsutani, V. Vorndam, M. Nakata, T.    Ayers, J. Elm, T. Torn, P. Reiter, J. G. Rigau-Perez, J. M.    Hayes, K. Mills, M. Napier, G. G. Clark, and D. J. Gubler. 2005.    Dengue fever, Hawaii, 2001-2002. Emerg Infect Dis 11:742-749.-   13. Franco, C., N. A. Hynes, N. Bouri, and D. A. Henderson. 2010.    The dengue threat to the United States. Biosecurity and    bioterrorism: biodefense strategy, practice, and science 8:273-276.-   14. Gomes, A. L., A. M. Silva, M. T. Cordeiro, G. F.    Guimaraes, E. T. Marques, Jr., and F. G. Abath. 2007. Single-tube    nested PCR using immobilized internal primers for the identification    of dengue virus serotypes. J Virol Methods 145:76-79.-   15. Gregory, C. J., L. M. Santiago, D. F. Arguello, E. Hunsperger,    and K. M. Tomashek. 2010. Clinical and laboratory features that    differentiate dengue from other febrile illnesses in an endemic    area—Puerto Rico, 2007-2008. Am J Trop Med Hyg 82:922-929.-   16. Gregory, C. J., and K. M. Tomashek. 2012. Management of severe    dengue. Pediatr Crit Care Med 13:125; author reply 125-126.-   17. Gubler, D. J. 2002. Epidemic dengue/dengue hemorrhagic fever as    a public health, social and economic problem in the 21st century.    Trends Microbiol 10:100-103.-   18. Gubler, D. J., G. Kuno, G. E. Sather, M. Velez, and A.    Oliver. 1984. Mosquito cell cultures and specific monoclonal    antibodies in surveillance for dengue viruses. Am J Trop Med Hyg    33:158-165.-   19. Gubler, D. J., W. Suharyono, I. Lubis, S. Eram, and S.    Gunarso. 1981. Epidemic dengue 3 in central Java, associated with    low viremia in man. Am J Trop Med Hyg 30:1094-1099.-   20. Henchal, E. A., S. L. Polo, V. Vorndam, C. Yaemsiri, B. L.    Innis, and C. H. Hoke. 1991. Sensitivity and specificity of a    universal primer set for the rapid diagnosis of dengue virus    infections by polymerase chain reaction and nucleic acid    hybridization. Am J Trop Med Hyg 45:418-428.-   21. Holmes, E. C., and S. S. Twiddy. 2003. The origin, emergence and    evolutionary genetics of dengue virus. Infect Genet Evol 3:19-28.-   22. Hsieh, C. J., and M. J. Chen. 2009. The commercial dengue NS1    antigen-capture ELISA may be superior to IgM detection, virus    isolation and RT-PCR for rapid laboratory diagnosis of acute dengue    infection based on a single serum sample. J Clin Virol 44:102.-   23. Huhtamo, E., E. Hasu, N.Y. Uzcategui, E. Erra, S, Nikkari, A.    Kantele, O. Vapalahti, and H. Piiparinen. 2010. Early diagnosis of    dengue in travelers: comparison of a novel real-time RT-PCR, NS1    antigen detection and serology. J Clin Virol 47:49-53.-   24. Johnson, B. W., B. J. Russell, and R. S. Lanciotti. 2005.    Serotype-specific detection of dengue viruses in a fourplex    real-time reverse transcriptase PCR assay. J Clin Microbiol    43:4977-4983.-   25. Lanciotti, R. S. 2003. Molecular amplification assays for the    detection of flaviviruses. Adv Virus Res 61:67-99.-   26. Leitmeyer, K. C., D. W. Vaughn, D. M. Watts, R. Salas, I.    Villalobos, C. de, C. Ramos, and R. Rico-Hesse. 1999. Dengue virus    structural differences that correlate with pathogenesis. J Virol    73:4738-4747.-   27. Low, J. G., A. Ong, L. K. Tan, S. Chaterji, A. Chow, W. Y.    Lim, K. W. Lee, R. Chua, C. R. Chua, S. W. Tan, Y. B. Cheung, M. L.    Hibberd, S. G. Vasudevan, L. C. Ng, Y. S. Leo, and E. E. Ooi. 2011.    The early clinical features of dengue in adults: challenges for    early clinical diagnosis. PLoS Negl Trop Dis 5:e1191.-   28. McElroy, K. L., G. A. Santiago, N. J. Lennon, B. W.    Birren, M. R. Henn, and J. L. Munoz-Jordan. 2011. Endurance, refuge,    and reemergence of dengue virus type 2, Puerto Rico, 1986-2007.    Emerg Infect Dis 17:64-71.-   29. Mohammed, H., M. Ramos, J. Armstrong, J. Munoz-Jordan, K. O.    Arnold-Lewis, A. Ayala, G. G. Clark, E. S. Tull, and M. E.    Beatty. 2010. An outbreak of dengue fever in St. Croix (US Virgin    Islands), 2005. PLoS One 5:e13729.-   30. Mohammed, H. P., M. M. Ramos, A. Rivera, M. Johansson, J. L.    Munoz-Jordan, W. Sun, and K. M. Tomashek. 2008. Travel-associated    dengue infections in the United States, 1996 to 2005. J Travel Med    17:8-14.-   31. Munoz-Jordan, J. L., C. S. Collins, E. Vergne, G. A.    Santiago, L. Petersen, W. Sun, and J. M. Linnen. 2009. Highly    sensitive detection of dengue virus nucleic acid in samples from    clinically ill patients. J Clin Microbial 47:927-931.-   32. Organization, W. H. 2009. Dengue guidelines for diagnosis,    treatment, prevention and control. Geneva    http://whqlibdoc.who.int/publications/2009/9789241547871_eng.pdf.-   33. Radke, E. G., C. J. Gregory, K. W. Kintziger, E. K.    Sauber-Schatz, E. A. Hunsperger, G. R. Gallagher, J. M.    Barber, B. J. Biggerstaff, D. R. Stanek, K. M. Tomashek, and C. G.    Blackmore. 2012. Dengue outbreak in key west, Florida, USA, 2009.    Emerg Infect Dis 18:135-137.-   34. Ramos, M. M., H. Mohammed, E. Zielinski-Gutierrez, M. H.    Hayden, J. L. Lopez, M. Fournier, A. R. Trujillo, R. Burton, J. M.    Brunkard, L. Anaya-Lopez, A. A. Banicki, P. K. Morales, B.    Smith, J. L. Munoz-Jordan, and S. H. Waterman. 2008. Epidemic Dengue    and Dengue Hemorrhagic Fever at the Texas-Mexico Border: Results of    a Household-based Seroepidemiologic Survey, December 2005. Am J Trop    Med Hyg 78:364-369.-   35. Rico-Hesse, R. 2007. Dengue virus evolution and virulence    models. Clin Infect Dis 44:1462-1466.-   36. Rico-Hesse, R. 1990. Molecular evolution and distribution of    dengue viruses type 1 and 2 in nature. Virology 174:479-493.-   37. Rigau-Perez, J. G., A. Ayala-Lopez, E. J. Garcia-Rivera, S. M.    Hudson, V. Vorndam, P. Reiter, M. P. Cano, and G. G. Clark. 2002.    The reappearance of dengue-3 and a subsequent dengue-4 and dengue-1    epidemic in Puerto Rico in 1998. Am J Trop Med Hyg 67:355-362.-   38. Sanchez-Seco, M. P., D. Rosario, L. Hernandez, C. Domingo, K.    Valdes, M. G. Guzman, and A. Tenorio. 2006. Detection and subtyping    of dengue 1-4 and yellow fever viruses by means of a multiplex    RT-nested-PCR using degenerated primers. Trop Med Int Health    11:1432-1441.-   39. Sharp, T. M., P. Pillai, E. Hunsperger, G. A. Santiago, T.    Anderson, T. Vap, J. Collinson, B. F. Buss, T. J. Safranek, M. J.    Sotir, E. S. Jentes, J. L. Munoz-Jordan, and D. F. Arguello. 2012. A    cluster of dengue cases in American missionaries returning from    Haiti, 2010. Am J Trap Med Hyg 86:16-22.-   40. Steel, A., D. J. Gubler, and S. N. Bennett. 2010. Natural    attenuation of dengue virus type-2 after a series of island    outbreaks: a retrospective phylogenetic study of events in the South    Pacific three decades ago. Virology 405:505-512.-   41. Thomas, L., V. Moravie, F. Besnier, R. Valentino, S.    Kaidomar, L. V. Coquet, F. Najioullah, F. Lengelle, R. Cesaire, A.    Cabie, and D. Working Group on. 2012. Clinical presentation of    dengue among patients admitted to the adult emergency department of    a tertiary care hospital in Martinique: implications for triage,    management, and reporting. Ann Emerg Med 59:42-50.-   42. Tomashek, K. M., A. Rivera, J. L. Munoz-Jordan, E.    Hunsperger, L. Santiago, O. Padro, E. Garcia, and W. Sun. 2009.    Description of a large island-wide outbreak of dengue in Puerto    Rico, 2007. Am J Trop Med Hyg 81:467-474.-   43. Tricou, V., N. N. Minh, J. Farrar, H. T. Tran, and C. P.    Simmons. 2011. Kinetics of viremia and NS1 antigenemia are shaped by    immune status and virus serotype in adults with dengue. PLoS Negl    Trop Dis 5:e1309.-   44. Twiddy, S. S., J. J. Farrar, N. Vinh Chau, B. Wills, E. A.    Gould, T. Gritsun, G. Lloyd, and E. C. Holmes. 2002. Phylogenetic    relationships and differential selection pressures among genotypes    of dengue-2 virus. Virology 298:63-72.-   45. World-Health-Organization. 2010. Impact of Dengue.    http://www.who.int/csr/disease/dengue/impact/en/index.html.

Patents and publications mentioned in the specification are indicativeof the levels of those skilled in the art to which the inventionpertains. These patents and publications are incorporated herein byreference to the same extent as if each individual application orpublication was specifically and individually incorporated herein byreference.

The foregoing description is illustrative of particular embodiments ofthe invention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

The invention claimed is:
 1. An isolated mixture of oligonucleotidescomprising the sequence of: SEQ ID NO: 3; SEQ ID NO: 6; or SEQ ID NO:12; at least one of the mixture further comprising a fluorescent label.